The focus of the Skeletal Biology Section is to biochemically characterize post-natal stem cells that form hard tissues (skeletal stem cells, to determine the factors that regulate their proliferation and differentiation, and to further define the hierarchy of stem cells, transiently amplifying progenitor cells, and differentiated cells that exist in hard tissues and their associated soft connective tissues. Skeletal stem cells are found amongst the adherent and clonogenic subset of bone marrow stromal cells and in peripheral blood. Based on studies conducted this year, it was determined that the proof of the existence of a skeletal stem cell is established through a complex sequence of ex vivo isolation and expansion, and in vivo transplantation. Through the ex vivo expansion of a single cell-derived strain, and the subsequent in vivo transplantation, a complete heterotopic bone/bone marrow organ, containing a hematopoiesis supporting stroma must be established in order to prove that the single, originally cloned cell was indeed a stem cell. In addition, stromal cells isolated from the heterotopic organ must be able to transfer the hematopoietic microenvironment and the potential to establish bone tissue in vivo, upon serial transplantation. It was found that in vivo transplantation of stromal cells from a variety of species usually does not lead to the formation of cartilage. This is due to the conditions of a relatively high oxygen tension established in open transplantation systems. Hence, the chondrogenic potential of the cell strain under examination must be separately probed using in vitro micromass cultures. Interestingly, however, it was found that certain clonogenic cells isolated from blood had the ability to form cartilage in micromass cultures, but did not have the ability to form bone in the in vitro transplantation assay. It was also determined that monolayers of stromal cells, established through clonal or non-clonal cultures, are not per se homogeneous populations of ?stem? cells, as is often stated in the literature, but an uncontrolled mixture of cells and progenitors of highly diverse differentiation potential. This is the natural consequence of the natural asymmetric kinetics of stromal stem cell growth in culture, which inherently leads to a progressive dilution of the original stem cells present in the explanted cells, unless some degree of expansion of the stem cells is allowed, during culture, through the stochastic reversal of asymmetric kinetics to symmetric expansion. From these studies, it is evident that in vitro and in vivo assays are necessarily complementary to one another in the study of the biology and pathology of skeletal stem cells, and cannot be sensibly used in isolation. Most in vitro assays of differentiation potential do not necessarily predict the behavior of the same test strain upon in vivo transplantation. Further studies are ungoing to develop a more complete finger-print of true stem cells, in order to be able to separate them from their more committed counterparts. In addition, studies utilizing human embryonic stem cells (hES, HSF6) were initiated in order to directly compare them to skeletal stem cells. The goal of this project is to determine what gene products are expressed by hES cells that support their extensive proliferation without differentiation. Furthermore, based on current knowledge of factors that regulate the predictable differentiation of skeletal stem cells into osteoblasts, chondrocytes, hematopoiesis-supportive stroma and adipocytes, attempts will be made to control the differentiation of hES cells into defined phenotypes.